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Biomedical subjects

P S Stewart

Publications and source records attributed to P S Stewart.

At least 19 recordsLinked to original sources
PubMed2005

Survival strategies of infectious biofilms.

Modern medicine is facing the spread of biofilm-related infections. Bacterial biofilms are difficult to detect in routine diagnostics and are inherently tolerant to host defenses and antibiotic therapies. In addition, biofilms facilitate the spread of antibiotic resistance by promoting horizontal gene transfer. We review current concepts of biofilm tolerance with special emphasis on the role of the biofilm matrix and the physiology of biofilm-embedded cells. The heterogeneity in metabolic and reproductive activity within a biofilm correlates with a non-uniform susceptibility of enclosed bacteria. Recent studies have documented similar heterogeneity in planktonic cultures. Nutritional starvation and high cell density, two key characteristics of biofilm physiology, also mediate antimicrobial tolerance in stationary-phase planktonic cultures. Advances in characterizing the role of stress response genes, quorum sensing and phase variation in stationary-phase planktonic cultures have shed new light on tolerance mechanisms within biofilm communities.

Adaptation, Physiological↗
PubMed2005

Ultrasonically controlled release of ciprofloxacin from self-assembled coatings on poly(2-hydroxyethyl methacrylate) hydrogels for Pseudomonas aeruginosa biofilm prevention.

Indwelling prostheses and subcutaneous delivery devices are now routinely and indispensably employed in medical practice. However, these same devices often provide a highly suitable surface for bacterial adhesion and colonization, resulting in the formation of complex, differentiated, and structured communities known as biofilms. The University of Washington Engineered Biomaterials group has developed a novel drug delivery polymer matrix consisting of a poly(2-hydroxyethyl methacrylate) hydrogel coated with ordered methylene chains that form an ultrasound-responsive coating. This system was able to retain the drug ciprofloxacin inside the polymer in the absence of ultrasound but showed significant drug release when low-intensity ultrasound was applied. To assess the potential of this controlled drug delivery system for the targeting of infectious biofilms, we monitored the accumulation of Pseudomonas aeruginosa biofilms grown on hydrogels with and without ciprofloxacin and with and without exposure to ultrasound (a 43-kHz ultrasonic bath for 20 min daily) in an in vitro flow cell study. Biofilm accumulation from confocal images was quantified and statistically compared by using COMSTAT biofilm analysis software. Biofilm accumulation on ciprofloxacin-loaded hydrogels with ultrasound-induced drug delivery was significantly reduced compared to the accumulation of biofilms grown in control experiments. The results of these studies may ultimately facilitate the future development of medical devices sensitive to external ultrasonic impulses and capable of treating or preventing biofilm growth via "on-demand" drug release.

Anti-Infective Agents↗
PubMed2002

Role of electrostatic interactions in cohesion of bacterial biofilms.

Significant decreases in the apparent viscosity of a bacterial biofilm suspension were measured following addition of sodium, potassium, magnesium, or calcium salts, whereas iron salts increased the viscosity. Electrostatic interactions contribute to biofilm cohesion and iron cations are potent crosslinkers of the biofilm matrix.

Biofilms↗
PubMed2002

Role of dose concentration in biocide efficacy against Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa entrapped in alginate gel beads to form artificial biofilms resisted killing by chlorine, glutaraldehyde, 2,2-dibromo-3-nitrilopropionamide (DBNPA), and an alkyl dimethyl benzyl ammonium compound (ADBAC). The degree of resistance was quantified by a resistance factor that compared killing times for biofilm and planktonic cells in response to the same concentration of antimicrobial agent. Resistance factors averaged 120 for chlorine, 34 for glutaraldehyde, 29 for DBNPA, and 1900 for ADBAC. In every case, resistance factors decreased with increasing concentration of the antimicrobial agent. An independent analysis of the concentration dependence of the apparent rates of killing of planktonic and biofilm bacteria showed that elevating the treatment concentration increased bacterial killing more in the biofilm than it did in a suspension culture. Calculation of a transport modulus comparing the rates of biocide reaction and diffusion suggested that at least part of the biofilm resistance to chlorine, glutaraldehdye, and DBNPA could be attributed to incomplete or slow penetration of these agents into the biofilm. Time-kill curves were nonlinear for biofilm bacteria in some cases. The shapes of these curves implicated retarded antimicrobial penetration for chlorine and glutaraldehyde and the presence of a tolerant subpopulation for DBNPA and ADBAC. The results indicate that treating biofilms with a concentrated dose of biocide is more effective than using prolonged doses of a lower concentration.

Biofilms↗
PubMed2002

Action of glutaraldehyde and nitrite against sulfate-reducing bacterial biofilms.

A continuous flow reactor system was developed to evaluate the efficacy of antimicrobial treatments against sulfate-reducing bacterial biofilms. An annular reactor operating at a nominal dilution rate of 0.5 h(-1) was fed one-tenth strength Postgate C medium diluted in 1.5% NaCl and was inoculated with a mixed culture enriched from oilfield-produced water on the same medium. Thin biofilms developed in this reactor after 2 days of operation. The activity of these biofilms resulted in approximately 50 mg S l(-1) of sulfide at steady state prior to biocide treatment. Biocide efficacy was quantified by recording the time required for sulfide production to recover following an antimicrobial treatment. In a control experiment in which pure water was applied, the time required to reach 10 mg S l(-1) sulfide after the treatment was 1.7+/-1.2 h, whereas the time to reach this level of sulfide after a pulse dose of 500 mg l(-1) glutaraldehyde was delayed to 61+/-11 h. Nitrite treatment suppressed sulfide production as long as the nitrite concentration remained above 15 mg N l(-1). Sulfide production recovered more rapidly after nitrite treatment than it did after glutaraldehyde treatment.

Anaerobiosis↗
PubMed2001

Antibiotic resistance of bacteria in biofilms.

Bacteria that adhere to implanted medical devices or damaged tissue can encase themselves in a hydrated matrix of polysaccharide and protein, and form a slimy layer known as a biofilm. Antibiotic resistance of bacteria in the biofilm mode of growth contributes to the chronicity of infections such as those associated with implanted medical devices. The mechanisms of resistance in biofilms are different from the now familiar plasmids, transposons, and mutations that confer innate resistance to individual bacterial cells. In biofilms, resistance seems to depend on multicellular strategies. We summarise the features of biofilm infections, review emerging mechanisms of resistance, and discuss potential therapies.

Bacteria↗
PubMed2001

Gene expression and protein levels of the stationary phase sigma factor, RpoS, in continuously-fed Pseudomonas aeruginosa biofilms.

Bacteria growing in biofilms experience gradients of environmental conditions, including varying levels of nutrients and oxygen. Therefore, bacteria within biofilms may enter distinct physiological states, depending on the surrounding conditions. In this study, rpoS expression and RpoS levels were measured as indicators of stationary phase growth within thick continuously-fed Pseudomonas aeruginosa biofilms. The level of rpoS expression in a 3-day-old biofilm was found to be three-fold higher than the average expression in stationary phase planktonic culture. RpoS levels in biofilms, indicated by immunoblot analysis, were similar to levels in stationary phase planktonic cultures. In planktonic cultures, oxygen limitation did not lead to increased levels of RpoS, suggesting that oxygen limitation was not the environmental signal causing increased expression of rpoS. These results suggest that bacteria within P. aeruginosa biofilms may exhibit stationary phase characteristics even when cultured in flow conditions that continually replenish nutrients.

Anaerobiosis↗
PubMed2001

Battling biofilms.

Explore the source record for details and available documents.

Bacterial Adhesion↗
PubMed2001

A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms.

AIMS: The purpose of this study was to develop a laboratory biofilm growth reactor system that simulated the toilet bowl environment and which could be used for biocide efficacy testing. METHODS AND RESULTS: A microbial biofilm reactor system incorporating intermittent flow and nutrient provision was designed. The reactor system was open to the air and was inoculated with organisms collected from toilet bowl biofilms. Once per hour, reactors were supplied with a nutrient solution for a period of 5 min, then flushed and refilled with tap water or tap water amended with chlorine. Quantitative measures of the rate and extent of biofilm accumulation were defined. Biofilm accumulated in untreated reactors to cell densities of 108 cfu cm-2 after approximately 1 week. Biofilm accumulation was also observed in reactors in the continuous presence of several milligrams per litre of free chlorine. Repeatability standard deviations for the selected efficacy measures were low, indicating high repeatability between experiments. Log reduction values of viable cell numbers were within ranges observed with standard suspension and hard surface disinfection tests. Biofilm accumulated in laboratory reactors approximately seven times faster than it did in actual toilet bowls. The same ranking was achieved in tests between laboratory biofilms and field-grown biofilms with three of the four measures, using three different concentrations of chlorine. CONCLUSION: This reactor system has been shown to simulate, in a repeatable way, the accumulation of bacterial biofilm that occurs in toilet bowls. The results demonstrate that this system can provide repeatable assays of the efficacy of chlorine against those biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The laboratory biofilm reactor system described herein can be used to evaluate potential antimicrobial and antifouling treatments for control of biofilm formation in toilet bowls.

Anti-Bacterial Agents↗
PubMed2001

Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates.

AIMS: The purpose of this study was to compare the efficacy, in terms of bacterial biofilm penetration and killing, of alkaline hypochlorite (pH 11) and chlorosulfamate (pH 5.5) formulations. METHODS AND RESULTS: Two species biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown by flowing a dilute medium over inclined stainless steel slides for 6 d. Microelectrode technology was used to measure concentration profiles of active chlorine species within the biofilms in response to treatment at a concentration of 1000 mg total chlorine l(-1). Chlorosulfamate formulations penetrated biofilms faster than did hypochlorite. The mean penetration time into approximately 1 mm-thick biofilms for chlorosulfamate (6 min) was only one-eighth as long as for the same concentration of hypochlorite (48 min). Chloride ion penetrated biofilms rapidly (5 min) with an effective diffusion coefficient in the biofilm that was close to the value for chloride in water. Biofilm bacteria were highly resistant to killing by both antimicrobial agents. Biofilms challenged with 1000 mg l(-1) alkaline hypochlorite or chlorosulfamate for 1 h experienced 0.85 and 1.3 log reductions in viable cell numbers, respectively. Similar treatment reduced viable numbers of planktonic bacteria to non-detectable levels (log reduction greater than 6) within 60 s. Aged planktonic and resuspended laboratory biofilm bacteria were just as susceptible to hypochlorite as fresh planktonic cells. CONCLUSION: Chlorosulfamate transport into biofilm was not retarded whereas hypochlorite transport clearly was retarded. Superior penetration by chlorosulfamate was hypothesized to be due to its lower capacity for reaction with constituents of the biofilm. Poor biofilm killing despite direct measurement of effective physical penetration of the antimicrobial agent into the biofilm demonstrates that bacteria in the biofilm are protected by some mechanism other than simple physical shielding by the biofilm matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This study lends support to the theory that the penetration of antimicrobial agents into microbial biofilms is controlled by the reactivity of the antimicrobial agent with biofilm components. The finding that chlorine-based biocides can penetrate, but fail to kill, bacteria in biofilms should motivate the search for other mechanisms of protection from killing by antimicrobial agents in biofilms.

Animals↗
PubMed2000

Modeling biofilm antimicrobial resistance.

A computer model capable of integrating mechanisms of biofilm resistance to disinfection by antimicrobial agents was developed. Resistance mechanisms considered included retarded penetration due to a stoichiometric reaction between the antimicrobial agent and biomass, incomplete penetration due to a catalytic reaction between the antimicrobial agent and the biomass, and the existence of a fraction of the cells in a resistant phenotypic state. Mathematical models of these processes were derived and solved in the computer simulation package MATLAB. Four sets of fitted experimental data on the disinfection of Pseudomonas aeruginosa biofilms were fit to each of the three models. No one model fit all of the data sets adequately. Killing of a 2-day old biofilm by tobramycin was best described by the physiological limitation model. Killing by hypochlorite was best described by the stoichiometric transport model. Killing by hydrogen peroxide was best simulated by the catalytic transport model. These results suggest that multiple mechanisms of biofilm reduced susceptibility are manifested even in biofilms of the same species and that the particular resistance mechanism depends on the biofilm age, antimicrobial agent, and biofilm thickness. The models presented in this article may be useful for diagnosing mechanisms of biofilm resistance from experimental data.

Biofilms↗
PubMed2000

Electrical enhancement of Streptococcus gordonii biofilm killing by gentamicin.

This electrical enhancement was demonstrated in an in vitro model. Streptococcus gordonii biofilms were grown for 6 days in continuous-flow reactors on one-tenth strength trypticase peptone broth. The biofilms attained a mean areal cell density of 2.4 x 10(8) c.f.u./cm2 and a thickness of approx. 19 microm. Biofilms exhibited characteristic resistance to killing by an antibiotic. When treated with 2 microg/ml gentamicin for 24 h, they exhibited a 0.84 log reduction in viable cell numbers; a 4.7 log reduction was measured in a planktonic culture. Killing of planktonic bacteria by this treatment was reduced to 1.2 log when an oxygen-scavenging enzyme was added to the medium. When a 2-mA direct current was applied during antibiotic treatment, biofilm killing increased to a 4.3 log reduction. Electrical current alone caused a 1.9 log reduction in biofilm cell counts. It is suggested that gentamicin was less effective against Strep. gordonii under anaerobic conditions than it was under aerobic conditions and that this can explain both the reduced susceptibility of the biofilm (due to oxygen depletion) and electrical enhancement of efficacy (due to oxygen generation by electrolysis).

Aerobiosis↗
PubMed2000

Reduced susceptibility of thin Pseudomonas aeruginosa biofilms to hydrogen peroxide and monochloramine.

Pseudomonas aeruginosa attached to alginate gel beads in sparse, thin biofilms exhibited reduced susceptibility to monochloramine and hydrogen peroxide compared with planktonic cells of the same micro-organism. Disinfection rate coefficients for planktonic bacteria averaged 0.551 mg(-1)min(-1) for monochloramine and 3.1 x 10(-4)l mg(-1) min(-1) for hydrogen peroxide. The corresponding values for 24-h-old biofilm cells were 0.291 mg min(-1) and 9.2 x 10(-5) 1 mg(-1) min(-1) for monochloramine and hydrogen peroxide, respectively. Several pieces of evidence support the interpretation that the reduced susceptibility of biofilm was not due simply to inadequate delivery of the antimicrobial agent to the local environment of the attached cells. No correlation between biofilm susceptibility and biofilm initial areal cell density was observed. Rapid delivery of hydrogen peroxide to the attachment surface, and subsequently to the interior, of the alginate gel beads was visualized by a direct experimental technique. Theoretical analysis of unsteady diffusion and diffusion reaction interactions also argued against any significant delay or barrier to antimicrobial or oxygen delivery. It was hypothesized that new genes are expressed when bacteria attach to a surface and begin to form a biofilm and that some of the resulting gene products reduce the susceptibility of the cell to antimicrobial agents including oxidative biocides such as monochloramine and hydrogen peroxide.

Alginates↗
PubMed2000

Role of RpoS and AlgT in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine.

The role of two sigma factors, AlgT and RpoS, in mediating Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine was investigated. Two knock out mutant strains, SS24 (rpoS-) and PAO6852 (algT-), were compared with a wild type, PAO1, in their susceptibility to monochloramine and hydrogen peroxide. When grown as biofilms on alginate gel beads (mean untreated areal cell density 3.7 +/- 0.27 log cfu cm-2) or on glass slides (mean untreated areal cell density 7.6 +/- 0.9 log cfu cm-2), wild type bacteria exhibited reduced susceptibility to both antimicrobial agents in comparison with suspended cells. On alginate gel beads, all strains were equally resistant to monochloramine. rpoS- and algT- gel bead biofilms of 24-hour-old were more susceptible to hydrogen peroxide disinfection than were biofilms formed by PAO1. Biofilm disinfection rate coefficients for the two mutant strains were statistically indistinguishable from planktonic disinfection rate coefficients, indicating complete loss of biofilm resistance. While 48-hour-old algT- biofilm cells became resistant to hydrogen peroxide, 48-hour-old rpoS- biofilm cells remained highly susceptible. With the thicker biofilms formed on glass coupons, all strains were equally resistant to both hydrogen peroxide and monochloramine. It is concluded that while RpoS and AlgT may play a transient role in protecting thin biofilms from hydrogen peroxide, these sigma factors do not mediate resistance to monochloramine and do not contribute significantly to the hydrogen peroxide resistance of thick biofilms.

Alginates↗
PubMed2000

Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin.

The penetration of two antibiotics, ampicillin and ciprofloxacin, through biofilms developed in an in vitro model system was investigated. The susceptibilities of biofilms and corresponding freely suspended bacteria to killing by the antibiotics were also measured. Biofilms of Klebsiella pneumoniae were developed on microporous membranes resting on agar nutrient medium. The susceptibilities of planktonic cultures and biofilms to 10 times the MIC were determined. Antibiotic penetration through biofilms was measured by assaying the concentration of antibiotic that diffused through the biofilm to an overlying filter disk. Parallel experiments were performed with a mutant K. pneumoniae strain in which beta-lactamase activity was eliminated. For wild-type K. pneumoniae grown in suspension culture, ampicillin and ciprofloxacin MICs were 500 and 0.18 microgram/ml, respectively. The log reductions in the number of CFU of planktonic wild-type bacteria after 4 h of treatment at 10 times the MIC were 4.43 +/- 0.33 and 4.14 +/- 0.33 for ampicillin and ciprofloxacin, respectively. Biofilms of the same strain were much less susceptible, yielding log reductions in the number of CFU of -0.06 +/- 0.06 and 1.02 +/- 0.04 for ampicillin and ciprofloxacin, respectively, for the same treatment. The number of CFU in the biofilms after 24 h of antibiotic exposure was not statistically different from the number after 4 h of treatment. Ampicillin did not penetrate wild-type K. pneumoniae biofilms, whereas ciprofloxacin and a nonreactive tracer (chloride ion) penetrated the biofilms quickly. The concentration of ciprofloxacin reached the MIC throughout the biofilm within 20 min. Ampicillin penetrated biofilms formed by a beta-lactamase-deficient mutant. However, the biofilms formed by this mutant were resistant to ampicillin treatment, exhibiting a 0.18 +/- 0.07 log reduction in the number of CFU after 4 h of exposure and a 1.64 +/- 0.33 log reduction in the number of CFU after 24 h of exposure. Poor penetration contributed to wild-type biofilm resistance to ampicillin but not to ciprofloxacin. The increased resistance of the wild-type strain to ciprofloxacin and the mutant strain to ampicillin and ciprofloxacin could not be accounted for by antibiotic inactivation or slow diffusion since these antibiotics fully penetrated the biofilms. These results suggest that some other resistance mechanism is involved for both agents.

Ampicillin↗
PubMed2000

Effect of catalase on hydrogen peroxide penetration into Pseudomonas aeruginosa biofilms.

The penetration of hydrogen peroxide into biofilms formed by wild-type and catalase-deficient Pseudomonas aeruginosa strains was measured using microelectrodes. A flowing stream of hydrogen peroxide (50 mM, 1 h) was unable to penetrate or kill wild-type biofilms but did penetrate and partially kill biofilms formed by an isogenic strain in which the katA gene was knocked out. Catalase protects aggregated bacteria by preventing full penetration of hydrogen peroxide into the biofilm.

Biofilms↗
PubMed1999

Bacterial biofilms: a common cause of persistent infections.

Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.

Anti-Bacterial Agents↗